1. Field of the Invention
The object of the present invention is a gel based electroanalytical microsensor and an integrated microsystem allowing reliable measurement of concentration for a variety of chemical compounds and ions in complex aqueous environment, avoiding interferences from other compounds present in the environment and allowing to distinguish between the different chemical forms of the compound of interest.
2. Background Art
Concentration of compounds in solution have long been determined by use of well-known electrodes combined with electrochemical and particularly dynamic electroanalytical techniques based on oxido-reduction processes. These techniques consist of recording current / potential curves, in other word functions F(U, I), under different experimental conditions. In voltammetric, polarographic and chronoamperometric techniques, the potential is swept as a function of time between two extreme values, and the resulting current is measured. In potentiometric techniques, the current is swept and the potential is recorded. In the present invention all these techniques can be used with the microsensor and the integrated microsystem, but for convenience for the rest of this description, only the term "voltammetry" will be used to represent the whole of these techniques. A variety of function F(U, I) have been proposed to carry out measurements in a large range of concentration and in particular to increase the sensitivity and the selectivity towards a given compound. The most commonly used voltammetric techniques are mentioned below. They allow the measurement of concentrations between 10.sup.-11 and 10.sup.-4 M (10.sup.-9 and 10.sup.-2 g/l), and they can be classified into the three following groups illustrated with non-restrictive examples.
i) Techniques without preconcentration of the test compound (analyte): Cyclic Voltammetry-CV. PA0 ii) Techniques with electrochemical preconcentration of the compound on the sensor: Anodic Stripping Voltammetry-ASV; Square Wave Anodic Stripping Voltammetry-SWASV. PA0 iii) Techniques with chemical preconcentration of the compound on the sensor: Adsorptive Cathodic Stripping Voltammetry-AdCSV.
In particular the techniques from the last two groups allow the detection of a large number of compounds at trace levels, as low as 10.sup.-11 M (in the order of 0.001 .mu.g/dm.sup.3). The said compounds can be organic or inorganic compounds under neutral, anionic or cationic form, such as metallic cations.
The electrodes traditionally used are usually made of gold, platinum or carbon and very often of mercury which presents the great advantage of surface reproducibility.
In "An Iridium based Mercury Film Electrode, part I, Selection of Substrate and Preparation" by S. P. Kounaves and J. Buffle (J. Electronal. Chem., 216, 53-69 (1987) Ref. 1), the authors showed that iridium is preferable to any other metal because of its high resistance to oxidation by oxygen, its low solubility in mercury and because of the good mercury-iridium cohesion. It is therefore possible to deposit mercury on the iridium, so that the electrode can be used either as an iridium electrode (without mercury), or as a mercury electrode, which extends its field of application. Classical electrodes can be found with various geometry and mechanical configurations (disc electrodes, rotating disc, hanging or dropping mercury drops, mercury film. . . ). Their dimensions are often in the range 0.1 mm to 10 mm. It is known that microelectrodes with dimensions less than 20 .mu.m present several advantages, such as the ability to work in quiescent or in highly resistive environments, as well as enhanced stability for the mercury films. The fabrication of mercury-plated iridium-based microelectrodes has been described in "A Mercury plated Iridium based microelectrode: preparation and some properties" by R. R. Vitre, M. L. Tercier, M. Tsacopoulos and J. Buffle (Anal. Chem. Acta 249, 419-425 (1991) Ref. 2).
Further improvements have been made in the fabrication procedure. For example S. P. Kounaves et al (patent U.S. Pat. No. 5, 378, 343, Ref. 3) used microtechnology for the fabrication of a microelectrode based sensor. In this patent, the working electrode is always made of mercury and is in direct contact with the aqueous solution to analyse.
Recently in "Fabrication and characterisation of mercury-plated iridium microelectrode arrays" by G. C. Fiaccabrino, M. L. Tercier, J. Buffle, N. de Rooij, M. Koudelpa-Hep (Transducers 95, Digest of Technical papers, vol. 2, 478 (1995), Ref. 4), the authors describe a fabrication procedure involving thin film technology and photolithography allowing the fabrication of a sensor analogous to the previous one, but with increased sensitivity and reliability.
Each single voltammetric technique allows to determine simultaneously the concentration of several compounds without modifying the solution where the measure is carried out. A given sensor allows thus to detect and to quantify a large number of organic or inorganic chemical compounds, which makes these techniques particularly useful for the chemical analysis of complex aqueous solutions of biological, industrial or environmental origins. These measurements can be carried out either after sampling, for example in a drop of blood, or directly in situ, for example in lake or sea waters and in sediments, by means of a probe including the sensor, or on line for example as detectors in separative chromatographic techniques or in flow injection analysis.
At present a number of difficulties limit the use of these techniques in complex environments. For routine analysis, it is necessary to make repetitive, accurate and reliable measurements over a long period of time, and the sensor should be able to work in hostile media and environment. The term "hostile media" includes solution to analyse containing compounds that we do not wish to detect but that can interfere with the measurement in a way or another by modifying the physicochemical conditions at the electrode surface, and in consequence the voltammetric signal. An example of this type of interference (interference 1) concerns compounds that are oxidised or reduced at the electrode surface with the compound to analyse and where oxidoreduction products react with the analyte. This is the case in particular with oxygen which is present in most of the solutions. Its reduction causes an increase of pH close to the electrode surface and eventually the precipitation of metal ions to analyse, as metal hydroxides. Another type of interference (interference 2) concerns macromolecules, colloids and other surface-active compounds which can adsorb at the electrode surface, and block or modify the electron transfer between the electrode and the compound to analyse. A third type of interference (interference 3) concerns contaminations, i. e. the release of ions to analyse, by the system itself. The term "hostile environment" includes the use of the microsensor in probes placed in situ in extreme conditions (bottom of oceans, sinking wells, industrial chemical reactors).
An additional limitation for the current sensors is the influence of the hydrodynamic flux or the stirring, on the measured signal. In particular for in situ measurements, where stirring is uncontrolled and can vary with time, this can lead to irreproducible results over long periods of time.
Finally, for the complex environments previously mentioned and particularly for metal ions, the compound to analyse is often present under different chemical forms (complexed or not, under various redox states, adsorbed or not on particles in suspension. . . ). For a correct analytical interpretation, the sensor should be capable of discriminating between the different forms.
In the presently known voltammetric sensors or electrochemical systems, the electrodes are directly in contact with the analytical environment. Such sensors or systems are not capable of solving the above mentioned problems.
One of the solutions used to solve the unreliability associated with the interferences of type 1 and 2 consists of drastically pretreating the samples by physical, chemical or biological means, which can eliminate the interferences (strong acids or oxidising agents, enzymatic attack. . . ). Such procedures have several disadvantages: large quantities of solvent are needed, they are time consuming, difficult if not impossible to use for in situ measurements, and errors can be generated for example by contaminations or loss by adsorption of the compound to analyse, from or on the pretreatment system.
Furthermore after a pretreatment, it is no longer possible to discriminate between the different chemical forms of the compounds to analyse, although it is often essential to know their relative content to solve industrial, medical or environmental problems.
The interfering compounds of type 2, which are the most frequently encountered in natural waters, biological and industrial fluids, are organic or inorganic colloidal substances or compounds with relatively high molecular mass, such as humic or fulvic compounds (vegetal decomposition products) present in soils and waters, as well as proteins or polysaccharides. Microporous membranes, for example made of Nafion or cellulose acetate have been proposed to protect the surface of the electrode. The efficiency and/or the reliability of preparation have shown to be poor and insufficient for voltammetric sensors which should be used in situ for long periods of time. Also in some cases, they cannot be used because they react with the test compounds.
None of the known techniques allow to satisfactorily protect the electrodes against hydrodynamic flow variations or stirring of the environment to analyse. In particular, reducing the size of the electrode is insufficient in itself to avoid hydrodynamic effects. Indeed these effects are negligible only for microelectrodes with size less than 0.2 .mu.m, whereas the smallest dimensions that can be obtained by the known photolithographic techniques are larger then 0.5 .mu.m. In this conditions, variations in the analytical signal of the order of 30 to 50% can be observed due to the influence of the hydrodynamic flow in the test solution.
Some limitations in the reproducibility of the measurements carried out with the prior art voltammetric sensors results from the said sensors fabrication itself. In particular limiting factors are the surface conditions of the deposited iridium film (which influences the cohesion of the mercury film on iridium), and the renewing conditions of the mercury films on the iridium. Indeed although a given film can be used for continuous measurements for at least several days, it must be renewed after this period of time. Adequate conditions must be used for this operation to avoid the formation of mercuric oxides at the surface of the electrode, leading to irreproducibility of the sensor. These aspects are not mentioned in the prior art patents (Ref. 3).
The present invention provides a sensor and an integrated electrochemical microsystem providing the appropriate conditions to avoid the corresponding problems. Anyway, it must be noticed that the absence of protection on the prior art voltammetric sensor is responsible for their fragility and their sensitivity to external conditions. The physical protection of the electrodes by the gel disclosed in the present invention greatly improves the long term stability of the sensors.